The Women's Health Initiative (WHI) evaluated the neurological costs and benefits of hormone therapy (HT) for post-menopausal women. Data from this study, citing negative effects of HT, differ from epidemiological and basic scientific evidence that suggests that estrogens are neuroprotective and neurotrophic, thereby enhancing some aspects of cognition. Despite the mean age for women experiencing menopause being 51 years (according to the Kaplan-Meier survival analysis), some women involved in the WHI study were, on average, 11-12 years post-menopause. When peri- and postmenopausal women are examined separately, the effects of HT differ significantly. It is thought that after circulating estrogens are nearly deplete for some time, re-exposure causes detrimental effects, an idea supported by the timing hypothesis4. This hypothesis suggests that there is a critical window of time during estrogen withdrawal that determines how reintroduction of estrogens will affect the body. Varied effects of HT reveal a missing link in our knowledge of the basic actions of estrogens, transmitted through receptors alpha and beta (ER-?. and ER?). Data from our laboratory reveal ligand-independent transcriptional actions of ???. Specifically, in the absence of estrogens, ER? activates transcription of target genes, including arginine vasopressin (AVP), a critical regulator of anxiety. Hence, this proposal is designed i) to elucidate specific regulatory actions modulating ER? during periods of estrogen withdrawal and ii) to determine how regulatory components effect anxiety regulation by ER?. To investigate these processes, Aim 1 will a) identify how hypoestrogenicity affects the conjugation of a transcriptional regulator, SUMO-1, to ER? and b) determine how SUMOylation alters ligand-independent transactivation of AVP and anxiety behavior. Aim 2 will a) identify coregulators associated with ER? during periods of hypoestrogenicity and then b) determine specific coregulators that are required for transactivation of AVP in the absence of ligand. To examine these issues, a battery of in vitro and in vivo experiments will employ cell culture and whole animal methodology. Two-dimensional SDS-PAGE and mass spectrometry will be performed to identify SUMOylation and coregulatory associations. To confirm SUMOylation of ER? and determine how this process affects transactivation of AVP, mutant receptors will be made and analyzed using reporter gene assays. The effect of SUMOylation on ER?-mediated anxiety behaviors will be assessed using behavioral anxiety tests. To confirm coregulatory interactions and determine specific coregulators for AVP transactivation, bioluminescence resonance energy transfer (BRET2), siRNA knockdown and reporter gene assays will be utilized. Overall, this proposal will reveal molecular mechanisms occurring during estrogen withdrawal that may alter the brain's receptivity to HT. PUBLIC HEALTH RELEVANCE: Hormone replacement therapy is a highly controversial but critical topic that affects women's health. This proposal aims to identify mechanisms that are responsible for dichotomous age-dependent effects of hormone replacement therapy during and after menopause.